Cvs transplantation for treatment of bacterial vaginosis

ABSTRACT

Methods and materials for treating bacterial vaginosis (“BV”) are provided. Cervicovaginal secretions (“CVS”) from a woman with vaginal microbiota dominated (&gt;50%) by one of the species of  lactobacillus  typically found in the human vagina, e.g.  Lactobacillus crispatus, L. iners, L. gasseri, L jensenii , is transplanted to women with BV as a method for restoring beneficial vaginal microbial communities and/or increasing resistance to sexually transmitted disease. Efficacy can be enhanced, or the properties of the endogenous CVS improved, through administration of an acidifying agent such as lactic acid. The examples demonstrate the role of healthy CVS in disease resistance, and the effect of pH on CVS properties. The examples also describe the collection and transplantation of healthy beneficial CVS into women at risk for, or after treatment for, BV.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International Applicationno. PCT/US2015/065002, filed on Dec. 10, 2015, which claims priority toand benefit of U.S. Provisional Application No. 62/091,970 “CVSTransplantation for Treatment of Bacterial Vaginosis” filed on Dec. 15,2014, the disclosures of which are hereby incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under NIH grantsR01HD062844, R21/R33A1094519, and R21/R33A1079740 by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention is generally in the field of treatment ofinfections of the vagina and sexually transmitted diseases.

BACKGROUND OF THE INVENTION

Approximately one-third of all women currently have bacterial vaginosis(BV), a condition where the vaginal microbiota is not dominated bylactobacilli. Another one-third of women have mixed vaginal microbiota(“intermediate BV”), and only one-third of women have healthy,lactobacilli-dominated microbiota. Women with BV are known tohave >2-6-fold increased susceptibility to numerous sexually transmittedinfections (STI), including HIV, herpes (HSV), gonorrhea, chlamydia, andother viral, bacterial, and protozoan pathogens. STI transmission ratesfrom women to men are higher if the woman has BV. Pregnant women with BVare much more susceptible to miscarriage, premature delivery, andpost-partum endometriosis. Strong links have also been establishedbetween BV and increased incidence of pelvic inflammatory disease andurinary tract infections.

Perhaps most alarming, few people have heard of BV, and even fewer knowhow to identify if they have it. The standard of care is vaginal or oralantibiotics. However, the effectiveness of antibiotics is limited bymutation leading to antibiotic-resistance, and estimates of BV relapse 4weeks after antibiotic treatment are as high as 70%. Although BV is anincredibly important global women's health issue, there is currently noknown long-term cure. Several attempts have been made to colonize thevagina with large doses of specific probiotic strains of lactobacilli,but the results have been disappointingly modest. In contrast toprobiotic strategies in which a single strain of dormant lactobacilli isplaced into an environment that is detrimental to its survival,isolating one important player in a complex mix of factors appears to betoo simplistic of an approach to be fully effective.

Studies have been conducted involving the introduction of probioticlactobacillus strains in isolation, which have demonstrated modestresults. One strain in particular, Lactobacillus crispatus CTV-05, hasbeen demonstrated to achieve colonization in the vaginas of womenwithout BV and was demonstrated as safe and tolerable in a Phase 2 trialin women with BV. Fecal transplants have been demonstrated to be safe,and have had as high as 94% effectiveness at eradicating C. difficileinfection in clinical studies. Probiotic products have also been used,as reported by Antonio M A, et al. J Infectious Dis 199(10); 1506-1513(2009), who studied microbial composition over time, and demonstratesthat pregnant women that have healthy, term pregnancies are more likelyto have Lactobacillus crispatus-dominated microbiota, and theirmicrobial communities are more stable over time. Romero et al.Microbiome, 2:4 http://www.microbiomejournal.com/content/2/1/4 (2014)was the first report of the temporal dynamics of vaginal microbiota inhealthy, reproductive age women. This paper discusses that Lactobacilluscrispatus dominated communities are more stable, and therefore, lessoften associated with transitions to a state of bacterial vaginosis(BV). Gajer et al. Science Translational Medicine, 4(132) 132ra52 (2012)demonstrated in pregnant women that L. crispatus colonization is morestable, and that L. iners is more conducive to the development of BV.Verstraelen et al. BMC Microbiology 9(116) (2009) describes the first,and now unethical, studies of BV-associated bacteria, with pregnantwomen. Gardnerella vaginalis alone was insufficient in initiating BV in12 out of 13 women with lactobacillus-dominated vaginal microbiota.However, 11 of 15 women inoculated with cervicovaginal fluid from womeninfected with G. vaginalis developed symptoms, indicating that otherenvironmental factors were needed for the bacteria to thrive. Criswellet al. Obstet and Gynecol. 33(2) 195-199 (1968) describes the prevalenceof BV amongst women who have sex with women. They found that of 58monogamous couples, 95% were concordant for the presence or absence ofBV, which was statistically very distinct from the normal distributionexpected in the female population. This would indicate that vaginalmicrobiota transfer must occur as a result of transfer of vaginalfluids. Marrazzo et al. JID. 185:1307-13 (2002) described BV relapserates of up to 70% one month after antibiotic treatment. See alsoLarsson and Forsum, APMIS. 113: 305-16 (2005).

The introduction of beneficial bacterial communities within theenvironmental milieu that supports their survival appears to be moreeffective than introducing isolated bacterial strains. No suchcommunities have been identified or tested for treatment of BV, however.

It is therefore an object of the present invention to provide a methodand materials for treating BV.

It is another object of the present invention to identify “donor”participants with the characteristics necessary for providing donorsamples for treatment and prevention of BV.

It is another object of the present invention to provide methods andmaterials for CVS transplants to increase the effectiveness of standardantibiotic treatments for treating bacterial vaginosis.

SUMMARY OF THE INVENTION

Method and materials for treating bacterial vaginosis (BV) are disclosedherein. Methods and materials for transplanting cervicovaginalsecretions (“CVS”) to increase the effectiveness of standard antibiotictreatments for BV are also described. Methods for identifying “donor”participants with the characteristics necessary for providing donorsamples for treatment and prevention of BV have also been developed.

Instead of isolating and purifying a particular strain, CVS from one ormore women with vaginal microbiota dominated (>50%) by species typicallyfound in the human vagina, e.g. Lactobacillus crispatus, Lactobacillusiners, Lactobacillus gasseri, Lactobacillus jensenii, is transplanted towomen with BV as a method for restoring beneficial vaginal microbialcommunities and/or increasing resistance to sexually transmitted diseaseand BV recurrence. Efficacy can be enhanced, or the properties of theendogenous CVS improved, through administration of an acidifying agent,before and/or after transplantation, such as lactic acid. The CVS canalso be filtered for sterility and to remove particles, aggregates andcells, for administration as a filtrate, and optionally mixed withisolated and cultured Lactobacillus bacteria, or spray dried orlyophilized and, optionally, packaged into single dosage unitapplicators.

The method is based on the following:

Only certain lactobacillus communities and strains are truly healthy andprotective against vaginal infection and should be transplanted to womenafter treatment for bacterial vaginosis.

The whole bacterial communities must be introduced in the vaginalenvironment to establish colonization, rather than isolated bacterialstrains.

Components of cervicovaginal mucus itself are beneficial to bacterialgrowth and survival, indicating that the mucus itself is the idealvehicle for introduction of microbiota into the vagina.

Vaginal microbiota transplantation can be supported by repeated vaginaldelivery of lactic acid or other similar compounds.

Examples demonstrate the role of healthy CVS in disease resistance, andthe effect of pH on CVS properties. The examples also describe thecollection and transplantation of healthy beneficial CVS into women atrisk for, or after treatment for, BV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs of the ensemble-averaged geometric meansquare displacements (<MSD>) as a function of time scale for (A) 1000 nmMPP (carboxylate-modified polystyrene nanoparticles with 2 kDapolyethylene glycol chemical conjugated to the surface to produce amucoinert surface coating), which diffuse similarly in CVS from womenwith healthy vaginal microbiota and from women with BV (FIG. 1A), and(B) HIV, which diffuses rapidly in BV CVS, but is immediately trapped inCVS from women with healthy microflora (FIG. 1B). W=diffusion rate inwater. FIG. 1C is a graph of the percentage of HIV with Log₁₀ (MSD)values at a time scale of 1 s. Minimal difference can be seen before andafter antibiotic treatment despite resolution of BV symptoms.

FIGS. 2A-2C are graphs. FIG. 2A is an abbreviated heat map showingrelative abundance of select bacterial species in CVS samples: group 1:L. crispatus-dominated; group 2: L. iners dominated; group 3: BVsamples. FIG. 2B is a plot illustrating that the percentage of D-lacticacid in CVS decreases as the percentage of L. iners (calculated in A)increases. FIG. 2C is a plot demonstrating that HIV mobility (score of5=100% mobility, score of 0=0% mobility) increases as the amount ofD-lactic acid decreases.

FIG. 3 is a graph of the survival of BV-associated bacteria in BV-CVS.Treatment with buffer or 1% lactic acid (LA) at pH 4.5 has minimaleffect on BV-associated bacteria. In contrast, 1% LA at pH 3.5(mimicking the healthy vagina) or the supernatant of a healthy L.crispatus-dominated CVS sample, resulted in 5-log reduction inBV-associated bacteria survival. In contrast, the L. iners-dominated CVSsample was far less effective (only ˜2-log reduction) at killingBV-associated bacteria in BV-CVS, and less effective than 1% LA alone.

FIGS. 4A-4H are graphs of the ensemble-averaged geometric mean squaredisplacements (<MSD>) as a function of time scale for HIV in CVS samplesthat do and do not trap HIV, and mixtures of trapping and non-trappingCVS samples. It was observed that mixing a non-trapping sample with ahealthy trapping sample leads to complete HIV trapping in the mixture,highlighting the ability of the healthy CVS to provide a beneficial pHenvironment for healthy microbiota, that also provides efficientpathogen trapping.

FIG. 5A is a bar graph showing numbers of colony forming units (CFUs, inlog scale) formed from BV-CVS incubated with saline or with cell-free,sterile-filtered CVS (CF-L-CVS) for two hours at 37° C. FIG. 5B is a bargraph showing reduction in the number of CFUs formed from BV-CVSincubated with undialyzed CF-L-CVS, CF-L-CVS dialyzed against salinewith a matched pH, or CF-L-CVS dialyzed against saline with a matched pHand lactic acid (LA) concentration, as compared to that formed fromBV-CVS incubated with saline. ** indicates p<0.01.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Probiotic, as used herein, utilizes the World Health Organization's 2001definition of “live micro-organisms which, when administered in adequateamounts, confer a health benefit on the host”. Probiotics must be alivewhen administered, have viability and reproducibility based on in vivotesting, and during use and storage.

Microbial flora refers to the microorganisms that normally live in thegastrointestinal tract, skin, nose, etc. In a healthy human, theinternal tissues, e.g. blood, brain, muscle, etc., are normally free ofmicroorganisms. However, the surface tissues, i.e., skin and mucousmembranes, are constantly in contact with environmental organisms andbecome readily colonized by various microbial species. The mixture oforganisms regularly found at any anatomical site is referred to as thenormal flora, except by researchers in the field who prefer the term“indigenous microbiota”. Bacteria are the most numerous microbialcomponents of the normal flora.

Microbiota, a term created by Jeffrey Gordon, refers to the collectionof microbial species that form a microbial community. This includes thenormal flora and “harmful” ones. “Microbiome,” refers to the collectionof genes present in the genomes of microbial species present in acommunity.

Bacterial vaginosis (“By”), as used herein, refers to the overgrowth ofone of several non-Lactobacillus types of bacteria normally present inthe vagina, upsetting the natural balance of vaginal bacteria.

Cervicovaginal secretions refers to the mixture of mucus secreted by thecervix, shed epithelial cells, vaginal transudate, and bacteria found inthe vagina of a woman.

Sexually transmitted diseases (“STD”) are any of various diseases orinfections (such as syphilis, gonorrhea, chlamydia, and genital herpes)that are usually transmitted by direct sexual contact and include some(as hepatitis B and AIDS) that may be contracted by other than sexualmeans.

As used herein, a “dissolution agent” is an acid, or salt thereof, thatis added to the vaginal area, vaginal secretions, cervicovaginalsecretions, or formulations containing cervicovaginal secretions. Thesecretions may be in dry (lyophilized) or wet form.

Cryoprotectant is any agent that prevents the formation of ice crystals,which can rupture cell membranes.

Diluent is any solution, optionally containing a cryoprotectant and/or adissolution agent. Diluent may optionally be balanced for a desiredosmolarity.

II. Formulations for Treatment of BV

A. CVS Transplants

The materials for transplant are secretions collected using standardtechniques from women with vaginal microbiota dominated (>50%) byspecies of lactobacillus typically found in the human vagina, e.g.Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri,Lactobacillus jensenii, who are healthy, free of sexually transmitteddisease and bacterial vaginosis, and have a low pH in the secretions. Asdescribed in Example 1, these can be collected using commerciallyavailable materials such as Instead Soft Cup menstrual fluid device,beaker, syringe, or absorbent matrix.

The secretions are preferably stored in the refrigerator at 4° C. for upto 1 week, or in certain cases, immediately frozen after collection andstored for up to several months, before being implanted into therecipient. Samples must maintain at least 20% viable bacteria prior touse.

The identity and relative abundance of bacteria in the CVS aredetermined by 16S rRNA pyrosequencing. The sequencing data is then usedto identify a community state, and only samples classified within thecommunity states of lactobacillus that are typically found in the humanvagina, including Lactobacillus crispatus, Lactobacillus iners,Lactobacillus gasseri, and Lactobacillus jensenii, will be consideredfor transplant. This community state often includes other species ofLactobacillus in smaller fractions. Genetic sequencing techniques andassignment of community states have been defined by the laboratory ofJacques Ravel at the University of Maryland.

B. Additives: Dissolution Agents, Cryoprotectants and Diluents

The secretions may be in dry (lyophilized) or wet form. Suitabledissolution agents include nitrogen-free organic acid having at leastone carboxylic acid group and a total of from 2 to about 20 carbonatoms, a phosphoric acid containing compound, a sulfonatedpolyphosphoric acid compound, a polyphosphonate having three or morephosphonate groups, an enzyme; or salts thereof; or combinationsthereof. Examples include lactic acid, citric acid, tartaric acid,gluconic acid, glycolic acid, hydroxysuccinic acid, galactaric acid,hydroxypropionic acid, lactic acid, glyceric acid, hydroxybutyric acid,hydroxyisobutyric acid, hydroxy methylbutyric acid, bis(hydroxymethyl)propionic acid, gibberellic acid, hydroxyoctadecanoic acid,di-tert-butyl hydroxybenzoic acid, benzilic acid, hydroxylfluorenecarboxylic acid, hydroxydecanoic acid,hydroxynaphthalenecarboxylic acid, hydroxybenzenedicarboxylic acid,hydroxymethylbenzoic acid, hydroxyphenylacetic acid, mandelic acid,hydroxymethoxybenzoic acid, methoxysalicylic acid, hydroxyoctanoic acid,hydroxy cinnamic acid, dihydroxycinnamic acid, dihydroxy-hydrocinnamicacid, hydroxyphenylpropionic acid, dihydroxytartaric acid,hydroxymethoxycinnamic acid, chlorohydroxybenzoic acid, chloromandelicacid, chloro phthalic acid, salicylic acid, chlorosalicylic acid,citrazinic acid, dibromo hydroxybenzoic acid, dichlorohydroxy-benzoicacid, dichlorosalicylic acid, galactouronic acid, glucuronic acid,hydroxypropanedioic acid, hydroxyphenyl propionic acid, lactic acid,methoxysalicylic acid, trihydroxybenzoic acid, or their partial saltsand combinations thereof. Another group of organic acids are varioushydroxyl free and nitrogen free saturated or unsaturated dicarboxylicacids having from 2 to about 20 carbon atoms and can contain nitrogenatoms. Examples include oxalic acid, malonic acid, succinic acid,glutaric acid, adipic acid, pimelic acid, maleic acid, fumaric acid,decanedoic acid, camphoric acid, benzenedicarboxylic acid, phthalicacid, cyclohexanedicarboxylic acid, cyclohexanediacetic acid, octanediocacid, homophthalic acid, phenylmalonic acid, cyclopentanediacetic acid,nonanedioic acid, benzylmalonic acid, phenylenediacetic acid,phenylsuccinic acid, bromosuccinic acid, carboxyphenacetic acid,cyclobutanedicarboxylic acid, cyclohexanedicarboxylic acid,decanedicarboxylic acid, dibromosuccinic acid, dichlorophthalic acid,diethylmalonic acid, diglycolic acid, dimethylmalonic acid, dimethylpentanedioic acid, dimethylsuccinic acid, ethylmalonic acid, glutamicacid, hexenedioic acid, imino diacetic acid, methylmalonic acid,methylsuccinic acid, naphthalene dicarboxylic acid, oxalacetic acid,oxopentanedioic acid, undecane dicarboxylic acid, dipicolinic acid, ortheir partial salts, and combinations thereof.

Cryoprotectant is any agent that prevents the formation of ice crystals,which can rupture cell membranes. Cryoprotectants include extracelluarcryoprotectants that do not penetrate bacterial cell walls, andintracellular cryoprotectants that penetrate bacterial cell walls.Examples of extracellular cryoprotectants include sucrose, dextrose andpolyvinylpyrrolidone (PVP). Examples of intracellular cryoprotectantsinclude dimethyl sulfoxide (DMSO) glycerol (glycerine).

Diluent is any solution, optionally containing a cryoprotectant and/or adissolution agent. Diluent may optionally be balanced for a desiredosmolarity. Exemplary diluent may be a solution of sodium chloride andlactic acid.

In a preferred embodiment, the recipient may also receive daily vaginaltreatment with a food acid such as a lactic acid gel, spray or powderbefore and/or after transplantation to encourage Lactobacillus growth.Daily treatment may occur for up to 1 week before and/or aftertransplantation. The preferred concentration range of lactic acid topromote Lactobacillus survival is 1-1.5% lactic acid. Lactic acid ispreferred to other types of food acid such as vinegar, lemon juice, andacetic acid, although these may also be utilized.

III. Methods of Treatment

A. Treatment of BV

Certain types of lactobacillus and compositions of vaginal microbialcommunities confer stability and resistance to bacterial vaginosis (BV).These are preferably obtained from women with vaginal microbiotadominated (>50%) by species of lactobacillus typically found in thehuman vagina, e.g. Lactobacillus crispatus, Lactobacillus iners,Lactobacillus gasseri, Lactobacillus jensenii, who are healthy, free ofsexually transmitted disease and bacterial vaginosis, and have a low pHin the secretions. These are administered to women with BV, asidentified clinically with Amsel's criteria, and confirmed in thelaboratory by Nugent scoring (Nugent et al., Journal of ClinicalMicrobiology, 29(2):297-301 (1991)). In a preferred embodiment, womenwith recurrent BV (requiring >3 treatment courses in 1 year) will firstbe treated with standard antibiotic treatment to reduce the bacterialload in the vagina. Twenty-four hours after the final antibiotic dose,the recipient will then receive a CVS transplant. The recipient willremain supine for at least 1 hour. Vaginal swabs will be collected fromthe recipient before transplant, after transplant, and at standardintervals (1 month, 2 months) after transplant. Characterization ofvaginal microbiota will be done by 1) Amsel's criteria, 2) Nugent score,and 3) 16S rRNA sequencing. “Success” will be defined as a lack of BVrelapse at 1 and 2 months after transplant, as assessed by Amsel'scriteria and Nugent score. 16S rRNA sequencing will reveal the relativeabundance of vaginal bacteria, and the degree of similarity to thecomposition of the transplanted sample.

B. Increased Resistance to STDs

CVS transplants also have barrier properties to sexually transmittedpathogens such as HIV are compromised in BV. However, these mucusbarrier properties do not appear to be restored, even after successfulantibiotic treatment, which is likely because the lactobacillus speciesmost commonly associated with BV (strains of L. iners) also have anegative impact on the vaginal mucus barrier. Transplantation of morebeneficial lactobacillus types, including the vaginal microbialcommunity and the mucus environment in which they live, is a promisingmethod for re-establishing healthy bacterial communities that do notcompromise the structural and adhesive properties of CVS in the vaginasof women with recurrent BV.

Several samples have been obtained with high Nugent scores and pH >4.5(together indicating BV) which look normal to the naked eye or are verythick in consistency, which may be a further reflection of the diversityof BV-associated microbiota and their effects on CVS. Changes in thelocal microstructure of BV samples, as probed by 1000 nm mucuspenetrating particles (MPP) (FIG. 1A), revealed a relative degradationof the CVS structure in BV. Additionally, due to the high pH (>4.5) ofBV-CVS, HIV diffuses rapidly in BV-CVS (FIG. 1B). BV-CVS has a similarlack of barrier properties to HSV. The effectiveness of treatment for BVand the barrier properties of CVS change after treatment withantibiotics. HIV rapidly diffused in CVS from women after antibiotictreatment, despite BV symptom (pH, consistency, odor) resolution,indicating that women that have ever been treated for BV may haveincreased susceptibility to STIs.

Although the CVS samples were acidic after treatment, it was found thatthe L/D lactic acid ratios were similar to when the participants had BV.This further supports the belief that certain strains of L. iners, thetype of lactobacilli most commonly present in the vagina after BVtreatment, may not be associated with increased protective benefits inthe vagina.

C. Dosage Unit Formulations

The CVS or a sterile filtered and pH adjusted CVS can be packaged intosingle dosage units for ease of administration. Typically these would bein a dispenser or applicator, sterile packaged, which has a tip forinsertion into the vagina, and a plunger to expel the packagedformulation.

In other embodiments, the CVS can be lyophilized or spray dried andstored frozen or in a sterile container, for resuspension immediatelyprior to use. The CVS can be resuspended with sterile water, a weakacidic solution, gel, or buffer.

In yet another embodiment, the spray dried formulation can be formulatedas a disk or wafer, which is inserted into the vagina where it hydratesand repopulates the vaginal mucosa.

In all of these embodiments, dyes, perfumes, pH buffering agents, dryingor resuspending agents, or other materials standard in the probioticfield can be incorporated into the formulations.

Typically, the dosage unit formulations contain between 10³ and 10¹⁵colony forming units (CFU) of any one of Lactobacillus crispatus,Lactobacillus iners, Lactobacillus gasseri, Lactobacillus jensenii. Insome aspects, the dosage units provide between 10³ and 10¹⁵ CFU of anycombination of Lactobacillus crispatus, Lactobacillus iners,Lactobacillus gasseri, Lactobacillus jensenii.

The present invention will be further understood by reference to thefollowing examples.

Example 1 Characterization of Mobility Differences in Normal and BVVaginal Mucosa

For CVS transplants to be successful, bacteria in the “donor” samplemust be in an environment that is beneficial to their survival.Lactobacilli thrive in an acidic environment with high lactic acidconcentration, which is inhospitable to many other types of bacteria,including those commonly associated with BV. Thus, it is important that,upon mixing with the vaginal secretions of a woman with BV, theenvironment must become acidified.

Materials and Methods

Ensemble-averaged geometric mean square displacements (<MSD>) as afunction of time scale for 1000 nm MPP, which diffuse more rapidly inCVS from women with BV than from women with healthy microbiota, and forHIV, were measured in BV-CVS as well as in CVS from women with healthymicrobiota (not on hormonal contraceptives). W=theoretical MPP/HIVdiffusion rate in water.

The Percentage of HIV with Log₁₀ (MSD) values at a time scale of 1 s inCVS from women collected before and after antibiotic treatment for BVwere calculated.

Results

FIG. 1 shows results obtained using multiple particle tracking (aquantitative fluorescent microscopy technique) and fluorescently labelednanoparticles or HIV virions. In FIG. 1A, the structural properties ofBV-CVS are investigated. The mucins in CVS normally form a mesh or netthat can sterically trap pathogens and particles. In BV-CVS, it wasobserved that non-adhesive nanoparticles (polyethylene glycol-coatedpolystyrene nanoparticles, termed mucus-penetrating particles or MPP) ofsimilar size to bacteria (1 μm in diameter) could more rapidly diffusein BV-CVS. Thus, FIG. 1A demonstrates the structural degradation of CVScaused by BV-associated bacteria.

FIG. 1B illustrates that BV-CVS also has reduced adhesive propertiescompared to CVS from women with healthy microbiota. HIV is small enoughin size (˜120 nm) to be able to diffuse through the pores in the mucinnet in CVS, yet it was observed that healthy CVS normally traps HIV.Thus, the interactions must be adhesive in nature. However, HIV diffusesrapidly in BV-CVS, indicating reduced adhesive interactions withpathogens. It is believed that BV-associated bacteria modify mucins(e.g. enzymatic cleavage of sugar binding sites), so they are no longeradhesive to pathogens.

FIG. 1C illustrates that the reduced adhesion of CVS to HIV virionsremains, even 1 month after antibiotic treatment for BV. It is believedthat the type of bacteria most likely to colonize the vagina after BV,Lactobacillus iners, also produces enzymes that modify mucins. Sampleswere obtained from numerous women that were previously treated for BVthat are L. iners-dominated, and HIV readily penetrates through theirCVS. It appears that only CVS transplants can recolonize the vagina withhealthy lactobacillus species, such as Lactobacillus crispatus. Even“successful” antibiotic treatment will lead to colonization by certainless-beneficial strains of L. iners, because it is the mainlactobacillus type where certain strains can survive the BV environment,whereas L. crispatus is typically not present after an episode of BV.

FIG. 2 shows that colonization by certain strains of L. iners, which isthe most likely after antibiotic treatment (if BV relapse does notoccur), is associated with CVS with impaired barrier properties. FIG. 2Ais an example of the heat map showing the relative abundance of bacteriain different CVS samples using 16S rRNA sequencing techniques (Dr.Jacques Ravel). As shown in FIG. 2B, a low concentration of D-lacticacid is associated with dominance by L. iners, which is then associatedwith increased mobility of HIV in the CVS. This further supports thehypothesis that even “successful” antibiotic treatment for BV (which mayoccur in only 50-70% of cases) is not enough to restore healthy barrierproperties in CVS. Thus, only CVS transplants can successfully restorethe most protective vaginal microbiota to women with BV.

Example 2 Killing of BV-Associated Bacteria in BV-CVS

Materials and Methods

Mixing experiments were performed to demonstrate killing ofBV-associated bacteria in freshly obtained BV-CVS. In these experiments,the “supernatant” (the fluid containing bacteria, soluble factors, etc.after centrifuging a CVS sample to separate the large molecular weightmucin components and cells) of healthy CVS samples was mixed withBV-CVS. The mixture was then plated to determine the effectiveness ofthe BV-associated bacteria “killing”.

Mixing experiments were performed in which CVS with pathogen trappingability was mixed 1:1 with CVS that did not trap pathogens (oneparticipant on hormonal contraceptives (HC), and one participant withBV).

For CVS transplants to be successful, bacteria in the “donor” samplemust be in an environment that is beneficial to their survival.Lactobacilli thrive in an acidic environment with high lactic acidconcentration, which is inhospitable to many other types of bacteria,including those commonly associated with BV. Thus, it is important that,upon mixing with the vaginal secretions of a woman with BV, theenvironment must become acidified.

Results

As shown in FIG. 3, buffer and 1% lactic acid (LA) at pH 4.5 (thevaginal pH cutoff commonly defined for BV) had little effect. Incontrast, 1% lactic acid at pH 3.5 (mimicking the healthy vaginalenvironment) was potent at inactivating BV-associated bacteria inBV-CVS. Similarly, the supernatant of an L. crispatus-dominated CVSsample produced a 5-log reduction in By-associated bacteria. Incontrast, the supernatant of an L. iners-dominated CVS sample was lesseffective than the supernatant from the L. crispatus sample or 1% LA atpH 3.5. This result supports two hypotheses: 1) healthy CVS, when mixedwith BV-CVS, can sufficiently alter the local environment to both killBV-associated bacteria and promote lactobacillus growth, and 2) L.crispatus-dominated CVS is much more potent than certain strains of L.iners-dominated CVS at killing BV-associated bacteria.

As shown in FIGS. 4A-4H, both CVS mixtures had moderate trapping abilityimmediately after mixing (MSD distribution shifted to the left, butstill a small percentage of rapidly diffusing HIV), likely because ofthe immediate drop in pH after mixing (compare values in Tables 1 and 2between starting samples and mixtures). The mixtures completely trappedthe virus after anaerobic incubation overnight.

TABLE 1 Mobility of HIV in CVS from healthy subjects, one hormonalcontraceptive receiving subject (HC), or one subject with BV. L/D SamplepH ratio HIV tracking 1 (HC) 4.34 1.6 mobile 2 (BV) 4.78 3.3 mobile 3(healthy) 3.93 0.8 trapped 4 (healthy) 3.80 1.0 trapped

TABLE 2 pH and L/D ratio stability in CVS mixtures obtained from mixingCVS of healthy subjects (3 or 4) with that of a hormonal contraceptivereceiving subject (HC) (1), or with that of a subject with BV (2). MixpH (10 min) pH (18 h) L/D ratio (18 h) 1 + 3 4.08 3.57 0.7 2 + 4 3.993.71 1.1

This experiment was conducted with 3 other sample combinations, withsimilar results: the pH of the CVS mixture immediately after mixing wasin the range ideal for lactobacillus growth, the CVS mixtures completelytrapped virus after incubation overnight, and the L/D ratio decreased toa level similar to the healthy CVS sample.

Example 3 Effects of Lactic Acid and pH from CVS with Lactobacilli onInactivating BV-Associated Bacteria

Materials and Methods

Production of Cell-Free Sterile-Filtered CVS (CF-L-CVS):

Whole CVS from individuals with lactobacilli was centrifuged at 20,000×gfor 2 minutes. The collected supernatant was then spun again over a 0.2micron spin filter.

Microdialysis:

CF-L-CVS was further pipetted into dialysis chambers and sealed with asquare of 1K MWCO dialysis membrane. Chambers were floated in a mediumwith one of the following dialysates for 2 hours, the dialysates beingsaline (normal saline, 0.9% sodium chloride, pH 7), saline (containing25 mM glycylglycine buffer and adjusted with hydrochloric acid andsodium hydroxide as needed) with pH to match the CF-L-CVS (sample pHranges ˜3-4), or saline (containing 25 mM glycylglycine buffer andadjusted with hydrochloric acid and sodium hydroxide as needed) withmatching pH and matching lactic concentration as the CF-L-CVS (sampleranges 0.5-1.5% lactic acid). Both saline buffers contained adjustedsodium chloride such that each buffer had the same osmolality. Thedialysis was sufficient to reduce the lactic acid concentration of CVSby ˜95%, unless the dialysate was supplemented with matchingconcentration of lactic acid.

BV-Associated Bacteria Inactivation Assay:

BV-CVS was diluted 1:100 into one of the following media, (i) saline,(ii) CF-L-CVS, (iii) CF-L-CVS that was dialyzed against saline, (iv)CF-L-CVS that was dialyzed against saline with matching pH as theCF-L-CVS, or (v) CF-L-CVS that was dialyzed against saline with matchingpH and matching lactic concentration as the CF-L-CVS. Diluted BV-CVS ina medium was incubated under anaerobic conditions at 37° C. before beingplated onto Brucella broth 5% sheep blood plates and incubatedanaerobically for 2-3 days, before colony forming units (CFUs) werecounted.

Results

As shown in FIG. 5A, incubation of BV-CVS with CF-L-CVS from individualswith lactobacilli for two hours greatly reduced the number of CFUscompared to incubating BV-CVS with saline. FIG. 5B shows that dialysisof CF-L-CVS against saline prevented its ability to inactivateBV-bacteria. Since dialysis against a lactic acid saline solutionretains the concentration of lactic acid in a dialyzed CVS and sincelactic acid is only a potent microbicide at an acidic pH, theconcentration of lactic acid and the pH of the lactic acid salinesolution were matched to those of the CF-L-CVS. Despite being dialyzed,retention of lactic acid in the CF-L-CVS inactivated BV-associatedbacteria similarly to undialyzed CF-L-CVS (FIG. 5B). Taken together,these results show that a high concentration of lactic acid in CVS fromindividuals with predominant lactobacilli microbiota inactivatesBV-associated bacteria in CVS from individuals with BV.

Example 4 Transplantation of Microbiota from Normal Vaginal Mucus

Approximately one-third of all women currently have BV, a conditionwhere the vaginal microbiota is not dominated by lactobacilli. Anotherone-third of women have mixed vaginal microbiota (“intermediate BV”),and only one-third of women have healthy, lactobacilli-dominatedmicrobiota. Women with BV have >2-6-fold increased susceptibility tonumerous sexually transmitted infections (STI), including HIV, herpes(HSV), gonorrhea, chlamydia, and other viral, bacterial, and protozoanpathogens. STI transmission rates from women to men are also higher ifthe woman has BV. Pregnant women with BV are much more susceptible tomiscarriage, premature delivery, and post-partum endometriosis. Stronglinks have also been established between BV and increased incidence ofpelvic inflammatory disease and urinary tract infections. The currentclinical treatment for BV includes vaginal or oral antibiotic treatment.Current experimental methods for treating BV include lactic acidgels/washes and vaginal probiotics, and have had minimal success.

A method for transplantation of CVS from a woman with healthy vaginalmicrobiota to a woman with BV as a means for restoring healthy vaginalmicrobiota has been developed. CVS transplants should be effective tocure or reduce symptoms or severity of BV, thereby having a major impacton women's sexual and reproductive health.

Materials

Donor participants will be recruited from the Johns Hopkins hospitalarea. They will be asked to provide CVS samples and vaginal swabs on theinitial visit for screening. If they are selected as a donor, the donorcandidate will be asked to return for a more thorough screening visitthat involves a routine gynecological exam, blood samples to screen forHIV and other infectious diseases, and vaginal swabs for sexuallytransmitted infection screening. Donor participants will agree toabstain from sexual contact between the screening visit and returning toprovide a CVS sample for donation to a recipient candidate. Donorparticipants, due to the nature of their CVS, are at a markedly reducedrisk of acquiring sexually transmitted infections.

CVS samples are obtained by insertion of an INSTEAD SOFT CUP® menstrualfluid device (as described in active protocols HIRB00000526,NA_00038105, and NA_00090758).

Methods

“Recipient” participants will be recruited from the Johns HopkinsOutpatient Center (JHOC). The recipients will be prescribed antibiotictreatment by a medical professional at the clinic. On the seventh(final) day of antibiotic treatment, the recipient will undergo aroutine gynecological exam, provide vaginal swab and CVS samples, andthen receive the CVS transplant.

Collected donor CVS samples are loaded into a syringe and inserted intothe vagina; similar procedures are used to administer and collect fluid(cervicovaginal lavage collection) as described in active protocolNA_00036496.

Control subjects receive a standard vaginal applicator loaded withvaginal placebo gel (hydroxyethylcellulose, HEC). The recipient will beasked to remain supine for 1 h after the procedure. After the procedure,the recipients will be counseled about the benefits of condom use andthe negative impacts of vaginal products on the success of thetransplant. The recipients will be given journals for logginginformation about sexual activity and vaginal product use, as well asswabs for taking twice weekly self-samples. The recipient will bescheduled to return for 1 month and 2 month follow-up visits andinstructed to bring their swab samples and journals to these visits. Thefollow up visits will involve a routine gynecological exam and the samesample collections as the initial visit and will take about 1 h.

Results

Definition of Treatment Failure or Participant Removal Criteria.

Donor participants will be removed from the study at any visit that theydo not fulfill the criteria (bacterial flora type, lack of sexuallytransmitted infections, no gynecological abnormalities, no high-risksexual behavior) for providing CVS donor samples. Recipients will not beremoved from the study if they do not follow the counseled suggestionsfor condom use, as the impact of sexual behavior is of interest for therelative success of the CVS transplants. Even if the recipients becomepregnant, the visits at month 1 and 2 are simply observational ratherthan interventional, and therefore do not pose a risk to the recipientparticipants. In the event that the recipients have BV relapse oracquire a sexually transmitted infection, they will be referred back tothe clinic where they were originally treated, and will be followedthrough the 2 month window. There is only 1 “dose” involved in the CVStransplant procedure. If the participants have a BV relapse or acquire asexually transmitted infection, they will be referred back to theirmedical provider at JHOC for treatment. CVS transplants are not intendedas an ongoing therapy at this time.

Primary Outcome Variable.

The primary outcome will be whether the recipient participations areclassified as having BV as assessed by standard Amsel's criteria in theclinic and by Nugent scoring at the 1 month and 2 month follow upvisits.

Secondary Outcome Variables.

The secondary outcome will be the composition of the recipientparticipant's vaginal microbiota at the 1 month and 2 month follow-upvisits, which will be determined by RNA analysis of the vaginal swabscollected.

The recipient participants can benefit by improved rates and longerduration of BV resolution, as standard antibiotic treatment often leadsto relapse within a month. As such, the women that receive only standardtreatment will likely have recurrent BV symptoms in addition to thenumerous sexual and reproductive risks associated with BV. In contrast,a successfully CVS transplant could provide prolonged resolution ofsymptoms and resistance to relapse.

Example 5 Long-Term Storage and Transplantation of CervicovaginalSecretions (CVS)

Materials

Cryoprotectant solution contains between 1% and 15% glycerol in saline.

Lactic acid-based diluent containing between about 0.5 and 1.5% lacticacid with sodium chloride and balanced to osmolality 200-300 mOsm, and apH of between 3.0 and 4.0.

Methods

1. Cervicovaginal secretions (CVS) will be collected whole andundiluted, generally yielding between 0.2 and 1 mL of material.

2. CVS will be slow frozen. Some samples will be mixed at up to 1:1dilution with cryoprotectant containing up to 15% glycerol and saline toimprove bacterial viability upon thawing. Ideal freezing conditions willbe evaluated for each “Donor's” CVS sample.

3. Upon thawing, CVS may be diluted up to a total volume of 5-50 mL witha lactic acid based diluent.

4. Diluted material may be incubated for up to 1 h at 37° C. to bringthe diluted CVS mixture up to body temperature.

5. Diluted CVS mixture may be administered to the vagina using a douchebottle with spray tip, a douche bottle, or other standard vaginalapplicator device.

“Donor” Identification

Donors will be screened to ensure:

1. Community dominance (>50% of overall bacterial community) of CVS byLactobacillus crispatus, iners, gasseri, jensenii or other relevantvaginal lactobacillus species;

2. Community dominance is stable (i.e. fluctuations occurring atmenstruation that recover back to similar community dominance throughoutthe next cycle, evaluated over 1-3 full menstrual cycles); and

3. CVS must have characteristics: pH <4, total lactic acid >0.8%, Nugentscore <2, negative by Amsel's criteria.

Freezing and Thawing

The CVS from various donors were collected and frozen at variousconditions described below.

The samples of frozen CVS were thawed by immersing sample tubes in 37°C. water bath for 10 minutes. The samples were then transferred toincubator set at 37° C. for 20 minutes, then diluted and plated.

Colony forming units of samples H260 and H257 (ID numbers denoteindividual patient samples) before freezing (Fresh), and after thawing,at a given dilution, were counted and presented in Tables 3-6 below.

Results

Data for freezing conditions suggest that each sample should beevaluated for optimal freezing conditions and use of a cryoprotectant.In some cases, CVS itself serves as the best cryoprotectant.

H260 (optimal conditions include 3.75% glycerol/saline with slowfreeze).

TABLE 3 Colony forming units in a fresh H260 sample diluted by a factorof 10⁸. 7.5% 3.75% 1.875% Dilution Fresh glycerol/saline glycerol/salineglycerol/saline 10⁸ 286 5 302 237

TABLE 4 Colony forming units in the H260 sample after thawing anddiluting by a factor of 10⁸. Slow Progressive 7.5% 3.75% 1.875% 1.875%freeze slow freeze glycerol/saline glycerol/saline glycerol/salineglycerol/saline Dilution 0 C. 0 C.; −15 C.; −80 C. fast freeze fastfreeze fast freeze slow freeze 10⁸ 4 0 0 114 334 620H257 (Optimal Conditions Appear to be Slow Freeze without Glycerol)

TABLE 5 Colony forming units in a fresh H257 sample diluted at indicateddilution factors. Dilution Fresh 7.5% glycerol 10⁶ >1000 600 10⁷ 878 32010⁸ 414 42

TABLE 6 Colony forming units in the H257 sample after thawing anddiluting at indicated dilution factors. 7.5% glycerol/saline fastDilution Slow freeze w/o glycerol freeze 10⁶ 936 720 10⁷ 481 457 10⁸ 224149

We claim:
 1. An isolated cervicovaginal secretion comprising vaginalmicrobiota, wherein at least 50% of the vaginal microbiota is onespecies typically found in the human vagina, e.g. Lactobacilluscrispatus, Lactobacillus iners, Lactobacillus gasseri, or Lactobacillusjensenii, having no evidence of sexually transmitted disease orbacterial vaginosis, an acidic pH less than 4.0.
 2. The isolatedcervicovaginal secretion of claim 1 which has been sterile filtered andpH adjusted.
 3. The sterile filtered and pH adjusted cervicovaginalsecretion of claim 2 containing Lactobacillus bacteria isolated, andoptionally cultured in vitro.
 4. The isolated cervicovaginal secretionof claim 1 packaged into a dosage unit or applicator for administrationto a human woman.
 5. The isolated cervicovaginal secretion of claim 1that has been frozen, optionally in combination with a cryoprotectant(i.e. glycerol), and thawed for administration to a human woman.
 6. Theisolated cervicovaginal secretion of claim 1 that has been mixed with adiluent containing up to 1.5% lactic acid, pH <4.0, for administrationto a human woman.
 7. The isolated cervicovaginal secretion of claim 1which has been spray dried or lyophilized and formulated foradministration to the vagina, optionally in combination with aresuspending or dissolution agent.
 8. A method for treating bacterialvaginosis comprising administering to the vagina an isolatedcervicovaginal secretion comprising vaginal microbiota, wherein at least50% of the vaginal microbiota is one species typically found in thehuman vagina, e.g. Lactobacillus crispatus, Lactobacillus iners,Lactobacillus gasseri, or Lactobacillus jensenii, having no evidence ofsexually transmitted disease or bacterial vaginosis, and an acidic pHless than 4.0, as defined by claim
 1. 9. The method of claim 8 furthercomprising subsequent daily vaginal administration of one to five mls ofa 1-1.5% lactic acid gel (pH <4.0) for up to one week before and/orafter transplantation of the isolated cervicovaginal secretion.
 10. Themethod of claim 8 wherein the bacterial vaginosis has been treated withantibiotics prior to administration of the cervicovaginal secretion. 11.A method for enhancing resistance to sexually transmitted diseasecomprising administering to the vagina an isolated cervicovaginalsecretion comprising vaginal microbiota, wherein at least 50% of thevaginal microbiota is one species typically found in the human vagina,e.g. Lactobacillus crispatus, Lactobacillus iners, Lactobacillusgasseri, or Lactobacillus jensenii, having no evidence of sexuallytransmitted disease or bacterial vaginosis, and an acidic pH less than4.0, as defined by claim
 1. 12. The method of claim 11 furthercomprising administering one to five mls of a 1 to 1.5% food acid gel(pH less than 4.0) daily for up to 1 week before and/or aftertransplantation of an isolated cervicovaginal secretion.